Nozzle assembly for a flow cytometry system and methods of manufacture

ABSTRACT

A method of manufacturing a nozzle assembly may include the step of over molding a nozzle housing, or a portion of a nozzle housing, onto at least one nozzle component, such as an injection tube. Nozzle assemblies and flow cytometers incorporating nozzle assemblies may include any combination of straight smooth injection tubes, improved features for securing a nozzle assembly, improved features for debubbling a nozzle assembly, and aggressive orienting geometries. A method of sorting cells may include the step of magnetically coupling a nozzle assembly with a flow cytometer.

This application is a national stage entry of International ApplicationNo. PCT/US2013/031787, filed Mar. 14, 2013, which claims priority toU.S. Provisional Application No. 61/703,102 filed Sep. 19, 2012, each ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of flow cytometryand more particularly to a nozzle assemblies for a flow cytometersystems and methods of manufacturing the same.

BACKGROUND

Flow cytometers have been known for analyzing and sorting particles, andare particularly suited to measure physical and chemical properties ofbiological materials, such as cells. In operation, a flow cytometerproduces a fluid stream which includes a sample fluid containingparticles of interest. These particles may be individually inspected inthe fluid stream with a variety of sensing systems or detection devicesand classified.

Sorters may additionally provide a mechanism for isolatingsubpopulations of particles based on their measured or determinedproperties. Jet-in-air flow cytometers achieve this separation throughthe creation and isolation of charged droplets containing particles ofinterest. The particle-containing droplets may be formed from the fluidstream and charged based upon a sort decision and, as they pass throughan electrical field produced by deflection plates, their path isredirected into one of several predetermined trajectories forcollection. The formation of these droplets may be achieved at a flowcytometer nozzle.

In addition to the function of droplet formation, a flow cytometernozzle may aide in biasing particles toward a uniform orientation. Thisfunction enables the analysis and sorting of cells which have asphericalproperties. In particular, the speeds at which sperm can be sorted intogender enriched populations have been increased, in part, due to thedevelopment of an orienting nozzle which presents a larger portion ofthe sperm in a relatively uniform orientation.

SUMMARY OF THE INVENTION

Certain embodiments of the claimed invention are summarized below. Theseembodiments are not intended to limit the scope of the claimedinvention, but rather serve as brief descriptions of possible forms ofthe invention. The invention may encompass a variety of forms whichdiffer from these summaries.

One embodiment relates to a method of manufacturing a nozzle assembly.Such a method may begin with the step of obtaining one or more nozzlecomponents and continue with molding one or more nozzle housing pieces,such that at least one nozzle housing piece is over molded onto at leastone of the nozzle components. Finally the nozzle housing pieces andnozzle components may be assembled into a nozzle assembly.

One embodiment provides for a nozzle assembly constructed from a nozzlehousing which encloses a nozzle cavity. A sample inlet may be formed inthe nozzle housing and connected to an injection tube having a sampleoutlet. The injection tube can be mounted to extend along the nozzlecavity and may include a flow path providing fluid communication betweenthe sample inlet and a sample outlet. A nozzle exit orifice may belocated downstream of the sample outlet and one or more sheath inletsmay be in fluid communication with the nozzle cavity.

Another embodiment provides a flow cytometer system that can include asample source supplying sample fluid containing particles of interest,as well as, a sheath source supplying sheath fluid to a nozzle assemblyfor forming a fluid stream. The nozzle assembly may produce a fluidstream along a flow path with sample and sheath fluid. The nozzleassembly may include a nozzle housing which encloses a nozzle cavity. Asample inlet may be formed in the nozzle housing. An injection tubehaving a sample outlet can be mounted with the nozzle housing and mayextend along the nozzle cavity. The injection tube can have a flow pathwhich provides fluid communication between the sample inlet and thesample outlet. The nozzle housing may further include one or more sheathinlets in fluid communication with the nozzle cavity and a nozzle exitorifice downstream of the sample outlet. The flow cytometer may furtherinclude an excitation source for interrogating particles within thefluid stream at an inspection zone and one or more detectors forproducing signals representative of emitted or reflected electromagneticradiation at the inspection zone. An analyzer may be included foranalyzing signals produced by the one or more detectors and for making asort decision. A charge element may charge the fluid stream according tothe sort decision for deflection by deflection plates.

Still another embodiment relates to a method of sorting cells which canbegin with the step of magnetically coupling a nozzle assembly having afluid flow path to a flow cytometer. Next a fluid stream may be formedat the nozzle assembly from a sheath fluid and a sample fluid. The fluidstream may be interrogated at an inspection zone which is then perturbedinto droplets. Signals representative of emitted or reflectedelectromagnetic radiation at the inspection zone may then be producedand analyzed for making sort decisions. Finally, droplets may beseparated according to the sort decision.

Yet another embodiment provides a nozzle assembly constructed from anozzle housing which encloses a nozzle cavity. An injection tube havinga sample outlet may be disposed within the nozzle cavity and the nozzlecavity may have an elliptical cross section with a major axis at leastthree times the length of the minor axis at the outlet of the injectiontube. One or more sheath inlets may be in fluid communication with thenozzle cavity and a nozzle exit orifice may be located downstream of thesample outlet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow chart of a process for producing a nozzleassembly for a flow cytometer.

FIG. 2 illustrates a sectional view of a flow cytometer system inaccordance with certain embodiments of the present invention.

FIG. 3 illustrates an exploded view of a nozzle assembly in accordancewith certain embodiments of the present invention.

FIG. 4 illustrates a sectional view of a nozzle assembly and other flowcytometer components in accordance with certain embodiments of thepresent invention.

FIG. 5 illustrates a closer sectional view of a portion of a flowcytometer nozzle assembly in accordance with certain embodiments of thepresent invention.

FIG. 6 illustrates a sectional view of a nozzle assembly in accordancewith certain embodiments of the present invention.

While the present invention may be embodied with various modificationsand alternative forms, specific embodiments are illustrated in thefigures and described herein by way of illustrative examples. It shouldbe understood the figures and detailed descriptions are not intended tolimit the scope of the invention to the particular form disclosed, butthat all modifications, alternatives, and equivalents falling within thespirit and scope of the claims are intended to be covered.

MODE(S) FOR CARRYING OUT THE INVENTION

A flow cytometer may include various individual components assembledinto suitable nozzles. The nozzle assemblies are tested to ensuredesired performance criteria is met. Such criteria can include whetherthe nozzle assemblies are providing sufficiently uniform orientation insome applications. Often nozzle assemblies fail to meet the desiredperformance criteria and are discarded or must be reworked.Occasionally, the position of an injection tube is not provided atprecisely the desired length or precisely within a desired radialposition, resulting in undesirable nozzle performance. Therefore, a needexists for a method of repeatedly and reproducible manufacturing nozzleshaving precise performance characteristics.

A certain aspect of the nozzle assembly described herein relates to astraight injection tube which is seated with a portion of the nozzleassembly. By reducing the overall length of the injection tube, itbecomes easier to control the length and radial position of theinjection outlet. Previous injection tubes often included metallicinjection tubes which were bent within the nozzle assembly or which werestraightened from coiled, or curved, stock. Whether introduced in apre-fabrication coiling step or just prior to deployment in a flowcytometer nozzle, such curvatures result in folds or irregularities onthe interior of the injection tube and may further create positionaluncertainty of the injection tube central axis with respect to thedesired flow axis within a nozzle. These folds and irregularities caninhibit laminar fluid flow or can redirect sample flow, which may have anegative impact on the performance characteristics of the nozzleassembly; particularly if orienting characteristics are desired. Inanother aspect, the over molded injection tube described herein maypresent a continuous, or flush, surface at any connection point. Variousprevious nozzle assemblies often included connectors which presenteddead volumes in the flow path. These dead volumes can become stagnantpockets of fluid that may harbor bacteria detrimental to the sample andmay be difficult to clean. By injection over molding an injection tubeinto the nozzle assembly a precise, repeatable length and position canbe achieved, thereby providing a reliable means of manufacturing nozzleassemblies with precise, reproducible performance characteristics.Additionally, over molding may provide a means for reducing oreliminating dead spaces at various connections. Additional elements maybe over molded, or injection molded, with various portions of the nozzleassembly to reduce the number of potential dead spaces as well as thenumber of connections with the potential for leaking.

Turning to FIG. 1, a flow chart illustrates a process for manufacturinga nozzle assembly for a flow cytometer. The process may begin at thestep generally designated “START” 1. Optionally, a portion of the nozzlehousing, or a nozzle assembly component, may be molded at the stepdesignated “MOLD” 2. By way of a non-limiting example, one of two piecesforming the nozzle housing may be molded at this step by an injectionmolding technique, such as by thermoplastic injection molding or bythermoset injection. In the case of thermoplastic injection molding, amold constructed from two or more cooperating sections forms a cavityhaving the shape of the nozzle housing piece. Molten resin can be fedinto that cavity under pressure and allowed to cool. Other moldingtechniques, such as transfer molding, compression molding,thermoforming, and other similar techniques may also be used to formportions of the nozzle housing. The molten resin may be selected frompolycarbonate, PVC, a plastic, polymer, plastomer, epoxy, phenolic, DAPand thermoplastics such as nylon, acetal, PBT, Polyphenylene oxide,Polyphenylene sulfide, or other similar materials. Although the step islabeled “MOLD,” it is also intended to encompass component parts made byfused deposition molding, such as in a 3-D printer (available fromStratasys, Edina, Minn., US).

At the step generally designated “INSERT MOLD” 3 a portion of the nozzlehousing may be injection molded, and further may be over molded onto acomponent of the nozzle assembly. By way of an example, a complimentarypiece of the nozzle housing may be formed for coupling with the firstpiece formed at the MOLD 2 step. Any one of, or any combination of, afirst component “component 1” 4 a, a second component “component 2”, athird component “component 3” 4 c, up to an nth component “component n”4 n, may be placed within the mold for over molding with a piece of thenozzle housing. This process may also be referred to as insert molding,by virtue of inserting a nozzle component into the mold. The nozzlecomponents which may be over molded into the nozzle housing may include:an injection tube, an oscillating element, a piezoelectric element, anozzle tip, a charge pin, an electrical cable, an electrical connector,a nozzle alignment mechanism, a particle alignment mechanism, a sheathinlet connector, a sheath inlet tube, a sample inlet connector, a wastetube, a metallic element, a ceramic element, an optical window, afastener, and a seating element. Any, or all, of the nozzle componentsmay be molded or manufactured contemporaneously with the stepsdescribed, or they may be pre-fabricated or even obtained from off theshelf sources. In one embodiment, an injection tube may be manufacturedby a polymer extrusion. Additionally, other components for nozzleassemblies may also be insert molded with a piece of the nozzle housing.In one embodiment, the initial step designated “MOLD” 2 may be skippedentirely and the INSERT MOLD 3 step may comprise molding a single piecenozzle housing. Such an embodiment may include over molding multipleadditional nozzle assembly components directly into the single mold. Byway of an illustrative example, an injection tube may be preciselyaligned and positioned within a mold at the INSERT MOLD 3 step. The moldmay be filled with molten resin to precisely, accurately andreproducibly provide the injection tube in a predetermined position in apiece of the nozzle housing. The insert tube may be obtained off theshelf, or may be fabricated, in the MOLD 2 step, and in one embodimentmay be constructed from a ceramic, a glass or a polymer. The injectiontube may include features for orienting particles, such as an externalgeometry, an internal geometry, an external bevel, or an internal taper.

The process may include additional iterations of the INSERT MOLD 3 stepfor insert molding additional nozzle components within additional piecesof the nozzle housing, or for making additional modifications to a pieceof the nozzle housing which was previously formed. As one example, thesecond piece of the nozzle housing may be over molded onto anoscillating element, such as a piezoelectric crystal. This step mayundergo any number of iterations and may be repeated any number of timesfor over molding additional pieces.

After any iteration of the INSERT MOLD 3 step, the process may continueto the step designated “ADDITIONAL MOLD,” 5 where additional pieces ofthe nozzle assembly or pieces of the nozzle housing may be molded. Inanother embodiment, the ADDITIONAL MOLD 5 step may be skipped. Thesepieces may be injection molded, transfer molded, compression molded,thermoformed, or formed with other similar techniques. The pieces may beformed from a polycarbonate, PVC, a plastic, or another polymer orplastomer. Additionally, any of these pieces may be produced by fuseddeposition molding, such as in a 3-D printer.

The process may continue by returning to the INSERT MOLD 3 step, forover molding additional pieces of the nozzle housing with various nozzlecomponents, or the process may continue to the step designated “POSTPROCESS” 6. In the POST PROCESS 6 step molded pieces may be finished andconnected. The POST PROCESS 6 may include washing, cleaning,sterilizing, curing, machining, and/or coating for any piece produced inthe MOLD 2, INSERT MOLD 3, and ADDITIONAL MOLD 5 steps. Additionally,these pieces may be fastened together, or provided with electrical,mechanical or fluid connections at the POST PROCESS 6 step. Followingthe POST PROCESS 6 step, the method may repeat iterations of the INSERTMOLD 3 or ADDITIONAL MOLD 5 steps. It should be appreciated, that FIG. 1illustrates a flow diagram, but that one or more of the steps may becarried out independently, or even at the same time.

As one example, a final iteration of the POST PROCESS 6 step may includethe steps of incorporating various nozzle components into one of twonozzle housing components, followed by coupling the housing pieces andcomponents into a nozzle assembly. The POST PROCESS step mayadditionally include various finishing processes, including, but notlimited to, glazing, sterilizing, chemical treatments, laser etching,laser detailing, or other post manufacturer processes. This nozzleassembly may be ready to receive fluid and electrical connections foroperation in a flow cytometer as designated “STOP” 7. In yet anotherembodiment, the POST PROCESS 6 may be skipped and the STOP 7 step may bereached after a final iteration of the INSERT MOLD 3 or ADDITIONAL MOLD5 steps. The final assembly may include a nozzle tip, or the nozzle tipmay be supplied later for flow cytometer operation. The processdescribed with respect to FIG. 1 may be incorporated for the manufactureof a large variety of nozzle designs, having a variety of potentialelements embedded within the nozzle housing pieces.

Turning now to FIG. 2, a flow cytometer system is illustrated whichincorporates one example of a nozzle assembly 10 that may bemanufactured by the process illustrated FIG. 1. The nozzle assembly 10may be incorporated at the sort head of any number of commerciallyavailable droplet sorters, such as jet-in-air flow cytometers. Thenozzle assembly 10 may include a nozzle housing 12 which encloses anozzle cavity 14. The nozzle housing 12 may be constructed from a singlemolded housing piece, or may be assembled from a collection of nozzlehousing pieces 44, such as two, three, four or more nozzle housingpieces. FIG. 2 illustrates a nozzle housing 10 which includes two nozzlehousing pieces 44 a, 44 b in the form of a nozzle cap 28 secured to anozzle base 30.

The flow cytometer system may include a sheath source 126 fluidicallycoupled to the nozzle assembly 10 for proving sheath fluid 128 to thenozzle assembly 10. A sample source 120 may also be coupled to thenozzle assembly 10 for providing sample fluid 122 to the nozzle assembly10. The sample fluid 122 and sheath fluid 128 may be introduced into anozzle cavity 14 under pressure and then passed through a nozzle tip 42having a nozzle exit orifice 26 to form a fluid steam 36 along a flowpath having a flow axis 94. The interior of the nozzle assembly 10 maybe configured for producing a fluid stream 36 from the nozzle exitorifice 26 in the form of coaxial stream having an inner core stream ofsample fluid 122 surrounded by an outer stream of sheath fluid 128.

An oscillating element 52, such as a piezoelectric crystal, may belocated within the nozzle assembly 10 for perturbing the fluid stream 36into droplets 60 some distance below the nozzle exit orifice 26.Previous oscillating elements have been located either above the nozzlecavity or within the nozzle cavity at the top of the cavity. One aspectof the current nozzle assembly 10 relates to an oscillating element 52which is positioned to surround a portion of the nozzle cavity 14 andreduces the distance between the oscillating element 52 and the nozzleexit orifice 26. The oscillating element 52 may have a ring or toroidalshape with an outer diameter and an inner diameter and may be incommunication with a controller 58. The controller 58 may produce adrive signal, such as between about 10 kHz and 120 kHz for perturbingthe fluid stream 36 into between about 20,000 droplets per second and120,000 droplets per second. Frequency and amplitude of the drive signalmay be manipulated and/or adjusted by a user through a graphic userinterface or through hardware. As but one example, the oscillatingelement 52 may be located about mid way down the nozzle assembly 10surrounding the nozzle cavity 14. This location may be within the nozzlehousing 12, or external to the nozzle housing 12, but mechanicallycoupled to the housing. Irrespective of the internal or externallocation, such an axial placement of the oscillating element 52 isbelieved to produce droplets more efficiently. In this configurationmechanical vibrations are transferred through nozzle assembly 10 andthrough the sheath fluid 128 in a speaker like manner to produce apulsing characteristic in the fluid stream 36. This pulsingcharacteristic eventually breaks the fluid stream 36 into droplets 60some distance below the nozzle exit orifice 26. Independent of otherinventive features described herein, this application contemplates thebenefit of modifying the placement of an oscillating element 52 withinor coupled to any nozzle for increased efficiency in producing droplets.

A charge pin 62 may be mounted with the nozzle assembly 10. The chargepin 62 may be constructed from any electrically conductive material andprovides an electrical connection between a charging element 52 andsheath fluid 128 contained in the nozzle cavity 14. Through the chargepin 62 a charge may be imparted to the entire fluid stream 36, includinga forming droplet just prior to breaking away from the fluid stream 36.An analyzer 178 or other processing device may determine physical orchemical characteristics of particles in the sample and may classify theparticles into one or more subpopulations. Based on any instructionsrelating to the subpopulation in which a particle is classified andother sorting parameters, including a calibrated drop delay, theanalyzer 178 will instruct a charge circuit 54 to charge the fluidstream 36 by charging the charge pin 62 just prior to the formation of adroplet in which that particle is expected. In this way, droplets 60 maybe supplied with a specific charge, including no charge, based on thecharacteristics of particles contained within them.

The nozzle assembly 10 may include a nozzle seat 102 for coupling intoposition on the flow cytometer system. Whereas previous nozzles may havebeen secured to adjustable stages with fasteners (such as screws, boltsetc.), the nozzle assembly 10 may include a nozzle seat 102 constructedfree from fasteners. As one example, the nozzle seat 102 may be coupledto a flow cytometer without the aid of fasteners.

An excitation source 130, such as a source of electromagnetic radiationmay be directed to a region know as an inspection zone 132 on the fluidstream 36. Particles within the fluid stream may reflect and/or emitelectromagnetic radiation in response to this excitation, and thisreflected and emitted electromagnetic radiation may be sensed by one ormore detectors 134. These detectors 134 may produce signalsrepresentative of the emitted or reflected electromagnetic radiation136, and those signals may be processed by an analyzer or a detectionsystem to derive a number of chemical and physical properties. Theanalyzer 178 may then provide instructions to the charge circuit 54 inorder to effect the appropriate sort action.

FIG. 3 illustrates an exploded view of a nozzle assembly 10. Such anozzle assembly 10 may be produced by a method such as the one describedin FIG. 1, or by another process. The exploded view illustrates a firstfastener 84 a and a second fastener 84 b for securing a first nozzlepiece 44 a, in the form of a nozzle cap 28, and a second nozzle piece 44b, in the form of a nozzle base 30, to a nozzle seat 102. The nozzleassembly 10 may, however, be constructed with any number of fasteners 84and nozzle pieces 44. In the illustrated embodiment, the nozzle seat 102includes a first threaded portion 82 a for receiving the first fastener84 a and a second threaded portion 82 b for receiving the secondfastener 84 b. In other embodiments the fasteners may be combined withand/or omitted in favor of adhesives, or other coupling means such asmagnets or mechanical means including springs.

The nozzle cap 28 may include a sample inlet 16 which is in fluidcommunication with an injection stem 32 and an injection tube 18 forforming a fluid flow path. The injection stem 32 may be integrallyformed with the nozzle cap 28, or they may be formed as separate nozzlepiece. The injection tube 18 may be over molded, or inset molded, withthe nozzle cap 28 in a manner which provides fluid communication betweenthe sample inlet 16 and the injection tube 18. This technique canprovide for a very short and precisely located injection tube 18. In oneembodiment a device may be coupled to the stem 32 which provides asurface with an adjustable axial position. As one example, the injectiontube 18 may be over molded onto such an element, which is thenmechanically coupled to the injection stem 32. In one embodiment, theinjection tube 18 is formed from a smooth rigid material to ensuredesired fluid flow properties. In an alternative embodiment, theinjection tube is formed from a more pliable material, which may bemanipulated after the injection tube is formed or molded. For example,the injection tube may be manipulated to change the initial geometry ofa fluid path formed there through for the purpose of encourage a ribboncore stream. As non-limiting example, modifications to the geometry maybe incorporated by laser etching certain portions or by a manufacturingstep of squeezing the injection tube while in pliable, and not perfectlyelastic state. Other manufacturing techniques may also be incorporatedto shape the outlet of the injection tube, such that one axis is longerthan a second axis. As but an illustrative example, other manufacturingtechniques may be employed resulting in an elliptical or rectangularinjection tube outlet.

The second nozzle piece 44 b, in the form of a nozzle base 30, may bedimensioned for coupling with the nozzle cap 28. An oscillating element52 may be insert molded with the nozzle base 30, or may be potted into acavity in the nozzle base 30. In one embodiment the nozzle base 30 isdimensioned to receive a nozzle tip 42. For example, the nozzle base 30may include interior dimensions for coupling with the nozzle tip 42,while the exterior of the nozzle base may be threaded for receiving aretaining nut 92 that holds the nozzle tip 42 in place. In anotherembodiment, the nozzle tip 42 may be insert molded with nozzle base 30,and in yet another embodiment the nozzle tip may be molded as a portionof the nozzle base 30.

The nozzle seat 102 may take the form of a nozzle clamp 78 whichreceives the first fastener 84 a and the second fastener 84 b in amanner which clamps the nozzle cap 28 to the nozzle base 30. The nozzleseat 102 may be dimensioned for fastener free coupling to the receiver150. As one example, the nozzle seat 102 can comprise a metallicmaterial coupled to a receiver 150 having magnetic properties. Amagnetic material may be located on either one of or both of the nozzleseat 102 and the receiver 150. In a similar embodiment, one or both ofthese components may be constructed to include electromagnets, ormaterials which demonstrate magnetic properties in response to electriccurrent. In this configuration, a nozzle assembly 10 may be simplydropped into place and held by gravity and the coupling of magneticcomponents. Such nozzles are quickly and easily interchangeable. In manyenvironments flow cytometer down time results in lost production timeand nozzles seat 102 as described herein provide an extremely efficientmethod of replacing nozzles and may improve the productivity of a givenflow cytometer system. The nozzle seat 102 and receiver 150 may beconstructed in a variety of other configuration for coupling the nozzleto a flow cytometer in a fastener free manner. In one embodiment thenozzle seat 102, or the receiver 150, may include springs for securingthe two pieces in a fastener free engagement. For example, a springloaded ball on one component may be designed to lock into socket on theother component. The nozzle seat 102 may also be physically dimensionedfor an interlocking configuration with a seat on an adjustable stage atthe flow cytometer head. In such an embodiment, the nozzle seat 102 maybe so dimensioned for being received by an adjustable stage. Once inplace, the nozzle seat 102 may be secured by rotation to achieve aninterlocking assembly, or by other mechanical means, such as mechanicalmeans provided on the adjustable stage.

The nozzle seat 102 may include an alignment element 154 in the form ofa protrusion which generally extends past a remaining boundary of thebottom surface of the nozzle seat 102. The receiver 150 may include analignment notch 152. The alignment element 154 and alignment notch 152may be so dimensioned to favor coupling in specified orientation. Inother embodiments, there may be a plurality of alignment notches 152 forpotentially securing a single alignment element 154. In thisconfiguration, the nozzle assembly 10 may rest in one of a plurality ofpredefined orientations relative to the flow cytometer system. Inanother embodiment, the receiver 150 is adjustable and may be secured ina plurality of positions for modifying the orientation provided byaligning the alignment element 152 and the alignment notch 154. In oneembodiment, a spring loaded ball may serve as both a means for engagingthe nozzle seat 102 with the receiver 105 and as the alignment element154 for aligning the two components. While additional components of theflow cytometer have not been illustrated, it should be understood thatthe receiver 150 may be firmly attached to a stage, such as a stagewhich is adjustable in two or three dimensions for alignment purposes.

The alignment element 154 and the alignment notch 152, in addition toproviding a specified orientation, may provide a precise nozzle locationallowing the rapid replacement of a nozzle assembly and minimizing theneed for realigning the flow cytometer. In combination with the magneticcoupling, this configuration may eliminate forces which tend to bringthe nozzle out of alignment with the detectors or source ofelectromagnetic radiation. Specifically, torque may be applied to theadjustable stage on which the nozzle sits when fasteners are securedinto place by the downward force an operator applies to the fastenersthemselves.

Grooves, slots, and other matched surfaces and geometries may also beused, alone, or in combination with magnetic coupling, to provideadditional configurations which allow the quick and precise matching toa preferred orientation and/or location. In another embodiment, visualaids in the form of marks or notches may be applied to the nozzle tofacilitate the quick and easy replacement of nozzles.

Turning now to FIG. 4, a more detailed sectional view of a nozzleassembly 10 is illustrated. The view illustrated in FIG. 3 is rotated ascompared to FIG. 2. The nozzle housing 12, comprising the nozzle cap 28and the nozzle base 30, along with the nozzle tip 42 form a nozzlecavity 14. The nozzle cavity 14 comprises an upper cavity 48 and a lowercavity 50. The upper cavity 48 may be considered the portion of thenozzle cavity 14 above the nozzle tip 42, while the lower cavity 50 maybe considered the portion of the nozzle cavity 14 formed at the nozzletip 42. The nozzle cap 28 and the nozzle base 30 may be held in positionby nozzle seat 102 in the form of a nozzle clamp 78 which receivesfasteners. A sealing element 118 may be provided at the contactingsurfaces of the nozzle cap 28 and the nozzle base 30. The sealingelement 118 may be an O-ring or another temporary, or permanent sealant.The nozzle tip 42 may be received by the nozzle base 30 and secured intoplace with a retaining nut 92. The nozzle tip 42 may have a taperingconical cross section, or may have an internal geometry designed toorient aspherical particles. Sperm orienting nozzle tips, like thosedescribed in U.S. Pat. Nos. 6,263,745, 7,371,517 and 8,206,988 arecontemplated for use with the described nozzle assembly 10 and eachpatent is incorporated herein by reference in its entirety. The nozzletip 42 may have a nozzle exit orifice 70 micrometers in diameter andsmaller or 60 micrometers in diameter and smaller. In alternativeembodiments, the nozzle assembly 10 may be formed from a singleinjection molded piece or from additional pieces. In an alternativeembodiment, the nozzle tip 42, nozzle base 30, and nozzle cap 28 areinjection molded to form a single piece nozzle assembly 10. In stillanother embodiment, the nozzle tip, nozzle base, nozzle cap, and even aninjection tube and/or injection stem may be produced as a single pieceby a fused deposition molding in a 3D printer. In such an embodiment,the nozzle tip may initially formed without an orifice, which may laterbe laser etched. Alternatively, a ceramic nozzle tip may be press fitinto a nozzle assembly produced in a 3-D printer.

One or more sheath inlets 24 a, 24 b provide fluid communication from anexterior surface of the nozzle housing 106 to the upper cavity 48 of thenozzle cavity 14. Specifically, the sheath inlets 24 connect a topsurface of the nozzle cap 180, which forms a portion of the exteriorsurface of the nozzle housing 106, to a bottom surface of the nozzle cap180, which forms a portion of the upper cavity 48. Each sheath inlet 24may be threaded for receiving a means to attach a sheath line.Alternatively, connectors may be formed integrally with the nozzle cap28 at each sheath inlet 24. Such integrated connectors may be formed byinjection molding the nozzle cap 28 and connectors as single piece.Additionally, a sample inlet 16 may be formed in the top surface of thenozzle cap 180 for communication with the nozzle cavity 14. In oneembodiment, tubing maybe directly coupled to the nozzle assembly 10 byover molding or through a secondary manufacturing process, such as byheating, glue or solvents, to provide a flush interior surface andreduce dead volume within the system. Such dead volume may require morecleaning and increases the potential for bacterial build up, leaking, aswell as the added expense and labor involved in more frequentreplacements.

The sample inlet 16 may continue through an injection stem 32. Theinjection stem 32 may be formed as a protrusion on the bottom surface ofthe nozzle cap 180 that extends into the nozzle cavity 14 and includes apocket 34 for receiving an injection tube 18. A flow path 20 within theinjection tube 18 may extend from the sample inlet 16 to a sample outlet22 disposed within the nozzle cavity 14. Pressurized sample fluid 122may be introduced through the injection tube 18 into the nozzle cavity14 and pressurized sheath fluid 128 may be introduced though the sheathinlets 24 a coaxial fluid stream 36 is formed at the sample outlet 22.The fluid steam 36 comprises an inner core stream of sample surroundedby an outer stream of sheath fluid. In certain applications relating toaspherical particles a greater degree of precision may be desirable inthe fluid mechanics of the injection tube because fluid mechanics andhydrodynamic forces may be used to attempt to bias the asphericalparticles toward a uniform orientation. Independent of other aspects ofthe disclosed nozzle assembly, an injection tube 18 may be molded from aceramic, molded or machined from glass, molded or machines from apolymer or other material to provide a smoother exterior surface 70 whencompared to injection tubes constructed from metal. Other additivefabrication (versus subtractive ie, milling/machining, turning)techniques may also be used to produce the injection needle 18. Typicalmetal injection tubes may include irregularities. In the case of bent orcurved injections tubes, irregularities such as folds may collect debriseffecting flow properties and providing bacteria an opportunity to grow.Another independent improvement in the current nozzle assembly is thatthe entire length of the injection tube and the entire sample flow pathwithin the nozzle assembly are located in a single flow axis 94 withoutcurves or bends. Such an arrangement may help the predictability ofhydrodynamic focusing in orienting nozzles and may help prevent thecollection of debris and the associated potential for bacterialinfection and clogging or reduced performance.

In one aspect, an injection tube 18 may be formed from a ceramicmaterial which may be molded into varied geometries. The interiorsurface of a ceramic injection tube can have a varied internal geometry40. FIG. 4 illustrates the varied internal geometry as an inward taper100 towards the sample outlet 22 of the injection tube 18. Other variedgeometries may be employed depending on the particles to be analyzed orsorted. For example, the interior geometry 40 could be an outward taper,as well as an elliptical or quadrilateral cross section. Othergeometries may also be used that are continuous, curved shapes,polygonal shapes, or geometries that contain curved and non-curvedshapes. Alternatively, the injection tube 18 may be produced from apolymer extrusion process, which may also result in a particularlysmooth interior surface.

The injection tube 18 may be incorporated into the pocket 34 of thenozzle cap 28 by injection over molding to ensure precise alignmentinjection tube 18 and precise location of the sample outlet. In oneembodiment, the injection tube 18 may be precisely seated in the nozzlecap 28 as a short straight tube which is glued into place. Additionalcomponents of the nozzle assembly 10 may be over molded or injectionmolded in order to reduce the number of seals and sealing elementsrequired. When the nozzle cap 28 is mated to the nozzle base 30, theinjection tube 18 is provided concentrically within the nozzle cavity 14with a high degree of precision. As illustrated in FIG. 4 the charge pin62 may be seen behind the sample inlet.

The nozzle cap 28 may also include a radial extension 148 that may serveas a grip for inserting and removing a fastener free nozzle assembly 10from a receiver 150. For example, the radial extension may provide agripping surface for separating the magnetically coupled components.

The oscillating element 52 may be potted in an outer cavity 184 of thenozzle base 30. The oscillating element 52 may be secured by any numberof methods and may even be injection molded directly into the nozzleassembly 10, or a portion of the nozzle assembly 10. The outer cavity184 may be separated from the nozzle cavity 14 by a uniform thickness ofmaterial in the nozzle base 30 along the length of the nozzle cavity 14.

Turning now to FIG. 5, sheath inlets 24 a, 24 b are illustrated in asectional view of the nozzle assembly 10 at the surface the nozzle base30 and the nozzle cap 28. The sheath inlets 24 a, 24 b may be designedto help prevent bubbles from lingering during or after various rinsingand cleaning cycles. Other surface treatments may be employed to furtherlessen air bubble entrapment during operation. For example, hydrophobicor hydrophilic treatments, may be applied either during or after nozzlefabrication. Such cycles may include pressurizing the first sheath inlet24 a while the second sheath inlet 24 b remains open to evacuate fluid.Flow may then be reversed by pressurizing the second sheath inlet 24 band releasing the first sheath inlet 24 a. Sheath flow may thus bemanipulated in a series of steps for varying durations to clean theinterior of the nozzle assembly 10. The turbulence and air introduced invarying the sheath flow during rinsing or cleaning can give rise tobubbles and certain embodiments of the present invention provide forde-bubbling features. A first de-bubbling counter sink 110 a may bepresent in the first sheath inlet 24 a at the nozzle cavity 14. Thiscountersink 110 a may provide for a first high point 108 a. Similarly, asecond de-bubbling counter sink 110 b may be present in the secondsheath inlet 24 b at the nozzle cavity 14, providing for a second highpoint 108 b. Bubbles that form in the nozzle cavity 14 will tend to riseto the high points 108 a, 108 b. Each de-bubbling counter sink 110 a,110 b can extend to the sealing element 118 to capture bubbles on theouter perimeter.

Turning now to FIG. 6, an embodiment is illustrated which may beconstructed through the same process described in FIG. 1. The nozzleassembly 310 may include a nozzle housing 312 constructed from a nozzlecap 328 and a nozzle base 330. The nozzle cap 328 may be constructedwith sample inlet 316 at a top surface. The sample inlet 316 maycontinue along a flow axis and be in fluid communication with a sampleoutlet 322 of an injection tube 318. The sample inlet 316 may providefluid communication through an injection tube 318 which terminates at anozzle tip 342.

The injection tube 318 may influence the flow of a sample fluid, forexample with a specified internal geometry 340, while the nozzle cavity314 geometry may influence the flow of sheath fluid. The sample andsheath are combined at the nozzle tip 342 and exit the nozzle assembly310 as a co-axial fluid stream. Unlike previous nozzle assemblies, theillustrated nozzle assembly 310 may provide a very aggressive initialtaper in its nozzle cavity 314. At the transition between the nozzlecavity 314 and nozzle tip 342, the cross sectional may have anelliptical cross section with a major axis three times longer than theminor axis. This geometry may promote a ribbon core flow, which may helpalign particles.

Various nozzle components may be incorporated in the nozzle housing 312through an insert or over molding manufacturing process, or they may beconnected to the nozzle assemblies in more conventional manner. Forexample, the nozzle cap 328 may be over molded onto a charge pin 362.FIG. 5 illustrates a conventional connection, which requires anadditional sealing element 354 to prevent leaking and to keep the nozzlecavity operating at a desired pressure.

Similarly, an oscillating element 352 may be potted into an outer cavity404 of the nozzle base 330. But it also may be over molded with thenozzle base 330, or even located on the exterior of the nozzle base 330.A seating element 402 may also be connected to the nozzle assembly 310.In one embodiment, the seating element comprises a clamp 378 whichreceives fasteners for securing the nozzle cap 328 to the nozzle base330. The seating element 402 or clamp 378 may be constructed from amaterial with magnetic properties, from a magnet, from an electromagnet,or may otherwise be designed for quickly and easily being secured intoplace on a flow cytometer.

As illustrated in FIG. 5 the nozzle tip 342 is held in place with aretaining nut 392, but the nozzle tip 342 may also be injection moldedas a portion of the nozzle housing 312. In a further alternativeembodiment, the nozzle tip 342 may be prefabricated and injection overmolded with the nozzle base 330.

As can be understood from the foregoing, various nozzle assemblyfeatures may be incorporated into a flow cytometer, as well as into amethod for manufacturing a flow cytometer. Those skilled in the art willrecognize that the invention described above includes many inventiveaspects, which may be provided in any combination and includes at leastthe following.

A1. A method of manufacturing a nozzle assembly comprising the steps of:a) obtaining one or more nozzle components; b) molding one or morenozzles housing pieces, wherein at least one nozzle housing piece isover molded onto at least one of the nozzle components; and c)assembling the nozzle housing pieces and nozzle components into a nozzleassembly.A2. The method of manufacturing a nozzle assembly as claimed in claim A1wherein the step of obtaining a nozzle component further comprises:obtaining an injection tube having a fluid flow path; and wherein thestep of molding one or more nozzle housing pieces further comprisesinjection molding a nozzle housing piece having a sample inlet, thenozzle housing piece having the sample outlet is over molded onto theinjection tube providing fluid communication between the sample inletand the fluid flow path.A3. The method of manufacturing a nozzle assembly as claimed in claim A2wherein the step of obtaining the injection tube comprises the step ofmolding an injection tube.A4. The method of manufacturing a nozzle assembly as claimed in claim A3wherein the injection tube is molded from a material selected from thegroup consisting of: ceramic, glass, and a polymer.A5. The method of manufacturing a nozzle assembly as claimed in claim A3wherein the injection tube is molded with features for orientingparticles.A6. The method of manufacturing a nozzle assembly as claimed in claim A5wherein the features for orienting particles include one or more of thefollowing: an interior geometry, an external geometry, an interiortaper, an external bevel.A7. The method of manufacturing a nozzle assembly as claimed in any oneof claims A2 to A6, further comprising the steps of: a) injectionmolding a first piece of the nozzle housing; b) over molding a secondpiece of the nozzle housing onto the injection tube; and c) securing thefirst piece of the nozzle housing with the second piece of the nozzlehousing.A8. The method of manufacturing a nozzle assembly as claimed in claimA7, wherein the second piece of the nozzle housing is injection moldedto form a nozzle cap having an injection stem.A9. The method of manufacturing a nozzle assembly as claimed in claimA8, wherein the nozzle cap is injection molded to include a sample inletand at least one sheath fluid inlet.A10. The method of manufacturing a nozzle assembly as claimed in claimA9, wherein the first piece of the nozzle housing is injection molded toform nozzle base which defines a nozzle cavity that receives theinjection tube.A11. The method of manufacturing a nozzle assembly as claimed in claimA10 further comprising the step of securing the nozzle cap to the nozzlebase such that the sheath inlet of the nozzle cap is in fluidcommunication with the nozzle cavity of the nozzle base.A12. The method of manufacturing a nozzle assembly as claimed in any oneof claims A1 to A10, wherein the injection tube is formed in a polymerextrusion.A13. The method of manufacturing a nozzle assembly as claimed in any oneof claims A1 to A12, further comprising the steps of obtainingadditional nozzle assembly components and securing those additionalnozzle assembly components with the nozzle assembly.A14. The method of manufacturing a nozzle assembly as claimed in claimA13 wherein the step of securing additional nozzle assembly componentswith the nozzle assembly further comprises over molding a portion of thenozzle housing.A15. The method of manufacturing a nozzle assembly as claimed in claimA14 wherein the nozzle components are selected from the group consistingof: an injection tube, an oscillating element, a piezoelectric element,a nozzle tip, a charge pin, an electrical cable, an electricalconnector, a nozzle alignment mechanism, a particle alignment mechanism,a sheath inlet connector, a sheath inlet tube, a sample inlet connector,a waste tube, a metallic element, a ceramic element, an optical window,a fastener, and a seating element.A16. The method of manufacturing a nozzle assembly as claimed any one ofclaims A1 to A15, further comprising the step of molding a nozzle tip,wherein the nozzle tip is dimensioned for coupling to the nozzlehousing.A17. The method of manufacturing a nozzle assembly as claimed in any oneof claims A1 to A17, wherein the step of injection molding a nozzlehousing further comprises the steps of injection molding the nozzlehousing as a single piece.B1. A nozzle assembly comprising: a) a nozzle housing enclosing a nozzlecavity; b)

a sample inlet formed in the nozzle housing; c) an injection tube havinga sample outlet, the injection tube being mounted with the nozzlehousing and extending along the nozzle cavity, wherein the injectiontube comprises a flow path providing fluid communication between thesample inlet and the sample outlet; d) one or more sheath inlets influid communication with the nozzle cavity; and e) a nozzle exit orificedownstream of the sample outlet.

B2. The nozzle assembly as claimed in claim B1, wherein the nozzlehousing comprises a nozzle cap and a nozzle base and wherein the sampleinlet is formed in the nozzle cap.

B3. The nozzle assembly as claimed in claim B2, wherein the nozzle capfurther comprises an injection stem disposed within the nozzle cavity,wherein the sample inlet extends through the injection stem.

B4. The nozzle assembly as claimed in claim B2 or B3, wherein the one ormore sheath inlets are formed in the nozzle cap.

B5. The nozzle assembly as claimed in claim B3 or B4, wherein theinjection stem further comprises a pocket and wherein the injection tubeis mounted with the pocket.

B6. The nozzle assembly as claimed in any one of claims B1 to B5,wherein the nozzle housing is injection molded with the injection tube.

B7. The nozzle assembly as claimed in any one of claims B1 to B5,wherein nozzle housing is over molded onto the injection tube.

B8. The nozzle assembly as claimed in any one of claims B1 to B7,wherein the injection tube comprises a ceramic injection tube.

B9. The nozzle assembly as claimed in claim B8, wherein the ceramic tubeinsert is beveled at the sample outlet.

B10. The nozzle assembly as claimed in claim B8 of B9, wherein the flowpath is at least partially defined by an interior geometry of theceramic injection tube.

B11. The nozzle assembly as claimed in claim B10, wherein the interiorgeometry of the ceramic injection tube is tapered inwardly towards thesample outlet.

B12. The nozzle assembly as claimed in claim B10 or B11, wherein theinternal geometry of the ceramic injection tube at, or towards, thesample outlet is selected from the group consisting of: an oval, asquare, a trapezoid, a rectangle, a cone, an inward taper, an outwardtape, and combinations thereof.B13. The nozzle assembly as claimed in any one of claims B1 to B12,further comprising a nozzle tip, wherein the nozzle exit orifice isformed in the nozzle tip.B14. The nozzle assembly as claimed in claim B13, wherein the nozzle tipcomprises an orienting nozzle tip.B15. The nozzle assembly as claimed in claim B13 or B14, wherein thenozzle tip is keyed such that the nozzle tip fits with the nozzlehousing in a specified orientation.B16. The nozzle assembly as claimed in any one of claims B13 to B15,wherein the nozzle exit orifice of the nozzle tip is 70 micrometers indiameter or less.B17. The nozzle assembly as claimed in any one of claims B13 to B15,wherein the nozzle exit orifice of the nozzle tip is about 60micrometers in diameter.B18. The nozzle assembly as claimed in any one of claims B1 to B17,wherein the nozzle cavity comprises an upper cavity formed by the nozzlehousing and a lower cavity formed by the nozzle tip.B19. The nozzle assembly as claimed in any one of claims B1 to B18,further comprising an oscillating element.B20. The nozzle assembly as claimed in claim B19, wherein saidoscillating element comprises a piezoelectric crystal mounted with thenozzle housing.B21. The nozzle assembly as claimed in claim B20, wherein theoscillating element has a ring shape and is disposed around a portion ofthe nozzle cavity.B22. The nozzle assembly as claimed in claim B20, wherein theoscillating element is disposed in an outer cavity.B23. The nozzle assembly as claimed in claim B20, wherein theoscillating element is mechanically coupled to the exterior of thenozzle housing.B24. The nozzle assembly as claimed in anyone of claims B19 to B23,wherein the nozzle assembly forms a fluid stream of sample and sheathfluid, when in use, which exits the nozzle exit orifice and wherein theoscillating element perturbs the fluid stream into droplets a distancebelow the nozzle exit orifice.B25. The nozzle assembly as claimed in claim B24, further comprising acharge pin for charging the fluid stream and droplets formed from thefluid stream.B26. The nozzle assembly as claimed in claim B25, wherein the charge pinis mounted with the nozzle cap.B27. The nozzle assembly as claimed in claim B25 or B26, wherein thecharge pin comprises a threading for threaded engagement with the nozzlecap.B28. The nozzle assembly as claimed in claim B25 or B26, wherein thecharge pin comprises a connector pin molded into the nozzle cap.B29. The nozzle assembly as claimed in any one of claims B1 to B28,further comprising a seating element mounted with the nozzle housing.B30. The nozzle assembly as claimed in claim B29, wherein the seatingelement comprises a material selected from a group consisting of: ametal, a magnetic material, an electromagnetic material, andcombinations thereof.B31. The nozzle assembly as claimed in claim B29 or B30, wherein theseating element is injection molded with the nozzle housing.B32. The nozzle assembly as claimed in claim B29 or B30, wherein theseating element comprises a nozzle clamp.B33. The nozzle assembly as claimed in claim B31, wherein the nozzleclamp comprises one or more threaded portions for receiving one or morefasteners to secure two or more portions of the nozzle housing.B34. The nozzle assembly as claimed in any one of claims B29 to B33,wherein the seating element is keyed to ensure the nozzle housing ismounted in a specified position or in a specified orientation.B35. The nozzle assembly as claimed in any one of claims B1 to B34,wherein the sample inlet is aligned coaxially with the sample outletalong a single flow axis.B36. The nozzle assembly as claimed in any one of claims B1 to B35,wherein the sheath inlet comprises more than one sheath inlet, eachsheath inlet being aligned in parallel with the flow axis.B37. The nozzle assembly as claimed in claim B36 wherein the flow axisis concentric with the nozzle exit orifice.B38. The nozzle assembly as claimed in any one of claims B1 to B37,wherein the length of the flow path between the sample inlet and thesample outlet is less than 50 mm.B39. The nozzle as claimed in any one of claims B1 to B37, wherein theinjection tube is formed from an extruded polymer.B40. The nozzle assembly as claimed in any one of claims B1 to B39,wherein the one or more sheath inlets further comprise:

-   -   a) a sheath port formed on the exterior surface of the nozzle        housing for receiving sheath fluid;    -   b) a debubbling countersink located on the interior surface of        the nozzle housing; and    -   c) a sheath inlet flow path connecting the sheath port to the        debubbling countersink.        B41. The nozzle assembly as claimed in claim B40, wherein the        debubbling countersink comprises a highpoint in the nozzle        cavity.        B42. The nozzle assembly as claimed in claim B40 or B41, wherein        the debubbling countersink extends outwardly to a sealing        element.        C1. A flow cytometer system comprising: a) a sample source        supplying sample fluid containing particles of interest; b) a        sheath source supplying sheath fluid; c) a nozzle assembly for        producing a fluid stream along a flow path, the fluid stream        having sheath fluid and sample fluid, wherein the nozzle        assembly comprises; i) a nozzle housing enclosing a nozzle        cavity; ii) a sample inlet formed in the nozzle housing; iii) an        injection tube having a sample outlet, the injection tube being        mounted with the nozzle housing and extending along the nozzle        cavity, wherein the injection tube comprises a flow path        providing fluid communication between the sample inlet and the        sample outlet; iv) one or more sheath inlets in fluid        communication with the nozzle cavity; and v) a nozzle exit        orifice downstream of the sample outlet; d) an excitation source        for interrogating particles within the fluid stream at an        inspection zone; e) one or more detectors for producing signals        representative of emitted or reflected electromagnetic radiation        at the inspection zone; f) an analyzer for analyzing signals        produced by the one or more detectors and for making a sort        decision; g) a charge element for charging the fluid stream        according to the sort decision; and h) deflection plates for        deflecting charged droplets to collection vessels.        C2. The flow cytometer system as claimed in claim C1, wherein        the nozzle assembly further comprises a seating element        constructed from a material selected from a group consisting of:        a metal, a magnetic material, an electromagnetic material, and        combinations thereof.        C3. The flow cytometer system as claimed in claim C2, wherein        the flow cytometer system further comprises a magnetic seat for        receiving the nozzle assembly.        C4. The flow cytometer system as claimed in claim C3, wherein        the seating element is keyed with an alignment notch and wherein        the magnetic seat comprises an alignment element for mating with        the alignment notch in a specified orientation.        C5. The flow cytometer system as claimed in any one of claims C1        to C4, wherein the flow path between the sample inlet and the        sample outlet is located entirely on a single flow axis.        C6. The flow cytometer system as claimed in claim C5, wherein        the injection tube is formed from an extruded polymer.        D1. A method of sorting cells comprising the steps of: a)        magnetically coupling a nozzle assembly having a fluid flow path        to flow cytometer; b) forming a fluid stream at the nozzle        assembly from a sheath fluid and a sample fluid; c) perturbing        the fluid stream into droplets; d) interrogating particles        within the fluid stream at an inspection zone; e) producing        signals representative of emitted or reflected electromagnetic        radiation at the inspection zone; f) analyzing the produced        signals for making sort decisions; and g) separating droplets        according to the sort decision.        D2. The method of sorting cells as claimed in claim D1, further        comprising the step of: a) removing the nozzle assembly from a        magnetic seat; and b) placing a replacement nozzle assembly in        the magnetic seat.        D3. The method of sorting cells as claimed in claim D2, further        comprising the steps of: a) aligning an alignment notch in the        replacement nozzle assembly with an alignment element in the        magnetic seat; and b) coupling the replacement nozzle assembly        to the magnetic seat.        D4. The method of sorting cells as claimed in claim D2 or D3,        wherein the replacement nozzle assembly is selected for its        fluid flow characteristics.        D5. The method of sorting cells as claimed in claim D4, wherein        the fluid flow characteristics are influenced by a distance        between a sample inlet and a sample outlet and/or an internal        geometry of a fluid flow path between the sample inlet and the        sample outlet.        D6. The method as claimed in any one of claims D1 to D5, further        comprising the step of cleaning the nozzle assembly.        D7. The method as claimed in claim D6, wherein the step of        cleaning the nozzle assembly further comprises the step of        debubbling the nozzle.        D8. The method as claimed in any one of claims D1 to D4, further        comprising the step of biasing particles in the fluid stream        towards a uniform orientation.        D9. The method as claimed in claim D8, further comprising the        step of aligning an orienting nozzle tip within the nozzle        assembly and orientating the nozzle assembly with respect to        detectors of the flow cytometer.        D10. The method as claimed in any one of claims D1 to D9,        wherein the step of separating droplets according to the sort        decision comprises separating a live X-chromosome bearing        subpopulation of sperm cells and/or a live Y-chromosome bearing        subpopulation of sperm cells from the remaining cells.        E1. A nozzle assembly comprising: a) a nozzle housing enclosing        a nozzle cavity; b) an injection tube having a sample outlet        disposed within the nozzle cavity, wherein the interior geometry        of the nozzle cavity at the sample outlet of the injection tube        comprises an elliptical cross section having a major axis at        least three times the length of the minor axis; c) one or more        sheath inlets in fluid communication with the nozzle cavity;        and d) a nozzle exit orifice downstream of the sample outlet.        E2. The nozzle assembly as claimed in claim E1, further        comprising an injection tube having an outer diameter less than        2 mm.        E3. The nozzle assembly as claimed in claim E1 or E2, wherein        the nozzle injection tube further comprises a geometry for        producing a ribbon core stream.        E4. The nozzle assembly as claimed in any one of claims E1 to        E3, wherein the nozzle assembly further comprises an alignment        feature.        E5. The nozzle assembly as claimed in any one of claims E1 to        E4, further comprising a nozzle tip.        E6. The nozzle assembly as claimed in claim E5, wherein the        nozzle tip comprises an internal geometry, wherein said internal        geometry begins as an elliptical cross section which tapers down        to a circular cross section moving down stream.        E7. The nozzle assembly as claimed in any one of claims E1 to        E6, wherein the nozzle cavity comprises an angle of taper        greater than 15 degrees.

As can be understood from the foregoing, the basic concepts of thepresent invention may be embodied in a variety of ways. As such, theparticular embodiments or elements of the invention disclosed by thedescription or shown in the figures accompanying this application arenot intended to be limiting, but rather exemplary of the numerous andvaried embodiments generically encompassed by the invention orequivalents encompassed with respect to any particular element thereof.In addition, the specific description of a single embodiment or elementof the invention may not explicitly describe all embodiments or elementspossible; many alternatives are implicitly disclosed by the descriptionand figures.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “acontainer” refers to one or more of the containers. As such, the terms“a” or “an”, “one or more” and “at least one” can be usedinterchangeably herein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes for example the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentapplication or continuation, division, or continuation-in-partapplication thereof, or to obtain any benefit of, reduction in feespursuant to, or to comply with the patent laws, rules, or regulations ofany country or treaty, and such content incorporated by reference shallsurvive during the entire pendency of this application including anysubsequent continuation, division, or continuation-in-part applicationthereof or any reissue or extension thereon.

The claims set forth in this specification, if any, are further intendedto describe the metes and bounds of a limited number of the preferredembodiments of the invention and are not to be construed as the broadestembodiment of the invention or a complete listing of embodiments of theinvention that may be claimed. The applicant does not waive any right todevelop further claims based upon the description set forth above as apart of any continuation, division, or continuation-in-part, or similarapplication.

We claim:
 1. A flow cytometer system comprising: a) a nozzle assemblythat receives a sample fluid containing particles and a sheath fluid,the nozzle assembly having an interior geometry that produces a fluidstream of the sheath fluid and the sample fluid along a fluid path, thenozzle assembly further comprising a nozzle seat and an orienting nozzletip secured in the nozzle assembly; b) a receiver that magneticallycouples to the nozzle seat, wherein at least one of the nozzle seat andthe receiver comprises a magnet; c) a rotational alignment elementformed on the nozzle seat, wherein the rotational alignment elementcomprises: a protrusion extending beyond a generally flat seatingsurface of the nozzle seat, grooves, or a surface configuration matchedto a surface of the receiver; d) an alignment notch formed in thereceiver that pairs with the rotational alignment element to secure thenozzle seat and the orienting nozzle tip in a predetermined rotationalorientation relative to the receiver when the nozzle seat and thereceiver are magnetically engaged and the rotational alignment elementand the alignment notch are coupled; e) an excitation source forinterrogating particles within the fluid stream at an inspection zone;f) one or more detectors for producing signals representative of emittedor reflected electromagnetic radiation at the inspection zone; and g) ananalyzer for analyzing the signals produced by the one or more detectorsand for making a sort decision.
 2. The flow cytometer system as claimedin claim 1, wherein the nozzle seat comprises a material selected from agroup consisting of: a metal, a magnet, a material having magneticproperties, a material having electromagnetic properties, andcombinations thereof.
 3. The flow cytometer system as claimed in claim1, wherein the receiver of the flow cytometer system comprises themagnet.
 4. The flow cytometer system as claimed in claim 1, wherein thenozzle assembly further comprises: a) a nozzle housing enclosing anozzle cavity; b) a sample inlet formed in the nozzle housing; c) aninjection tube having a sample outlet, the injection tube being mountedwith the nozzle housing and extending along the nozzle cavity, whereinthe injection tube comprises a flow path providing fluid communicationbetween the sample inlet and the sample outlet; d) one or more sheathinlets in fluid communication with the nozzle cavity; and e) a nozzleexit orifice downstream of the sample outlet.
 5. The flow cytometersystem as claimed in claim 4, wherein the injection tube has an outerdiameter less than 2 mm.
 6. The flow cytometer system as claimed inclaim 4, wherein the nozzle assembly injection tube further comprises ageometry for producing a ribbon core stream.
 7. The flow cytometersystem as claimed in claim 4, wherein the flow path between the sampleinlet and the sample outlet is located on a single flow axis.
 8. Theflow cytometer system as claimed in claim 7, wherein the orientingnozzle tip comprises an internal geometry, wherein said internalgeometry begins as an elliptical cross section which tapers down to acircular cross section along the single flow axis.
 9. The flow cytometersystem as claimed in claim 4, wherein the injection tube is formed froman extruded polymer.
 10. The flow cytometer system as claimed in claim4, wherein the nozzle cavity comprises an angle of taper greater than 15degrees.